Assembly of the large bacterial ribosomal subunit (50S) requires the coordinated synthesis of two rRNAs (5S and 23S) and 33 ribosomal proteins (r-proteins), processing and modification of the rRNAs, and assembly of both rRNAs and r-proteins into the functional subunit. Although assembly of the 50S subunit can be reconstituted in vitro, ribosome biogenesis in vivo has not been described. Since in vivo ribosome assembly also requires the activities of a number of ribosome assembly factors, determining when these assembly factors bind and defining the pre-50S particles that accumulate in their absence is critical for understanding in vivo assembly. Our long-term goal is to delineate the cellular events that lead to the generation of the 50S subunit. We are particularly focused on solving the temporal changes that occur during 50S biogenesis and the roles of ribosome assembly factors. We are now well poised to achieve this goal having recently made significant advances in defining the in vivo 50S particle using advanced proteomic approaches. Moreover, we have successfully used cutting edge proteomics to analyze one pre-50S intermediate. We have in hand a large number of verified ribosome-associated proteins and ribosome-assembly factors. We also have the genetic reagents (mutants, cloned genes and protein fusions) to define the assembly pathway for this complicated macromolecular structure. In this proposal, we will provide detailed characterization of assembly intermediates, isolate additional putative assembly factors and define functional relationships between ribosome-associated proteins and r-proteins. In Aim 1, we will define the in vivo assembly pathway for the 50S ribosomal subunit. To do this, we will build upon existing tools and reagents, guided in large part by the elegant ribosome intermediate studies being performed in S. cerevisiae. Our approach will be to purify intermediates in ribosome assembly and characterize both the rRNA and proteins. In these studies, we will apply proteomic approaches (iTRAQ quantitation technology) to define ribosome assembly intermediates. In addition to characterizing particles affinity purified pre-50S complexes from wild type cells, we will define intermediates that accumulate in a variety of ribosome assembly mutants. New proteins identified in this study will be assessed for their involvement in 50S assembly. In Aim 2, we will identify heretofore-unknown relationships by isolating high copy suppressors of existing ribosome assembly mutants. We will also define the relationship between the suppressor and the suppressed gene at the level of ribosome assembly and also in regards to testing specific predictions that we will develop for each suppressed mutant-suppressor pair. Experiments to test specific hypotheses concerning existing genetically interacting proteins are proposed. Project Narrative: Ribosomes are the largest macromolecular complex in the cell and, in bacteria, are the target of many antibiotics. Although the atomic structure of ribosomes has been solved and an in vitro assembly pathway defined, we know little about how ribosomes are assembled inside of bacterial cells. The goal of this proposal is to define the in vivo assembly pathway of the bacterial large ribosomal subunit, a feat that will have significant implications on the design of new antimicrobials and on understanding human diseases associated with the conserved mitochondrial ribosomes.